Detection of creatine levels using enzyme compositions

Abstract

The invention provides compositions and systems that allow the sensitive determination of the level of creatinine in a particular solution. Through the optimisation of enzymatic methods to detect creatinine the real-time determination of creatinine levels and creatinine clearance rates are also provided, allowing the real-time monitoring of kidney function. This is considered to be useful both in the monitoring of live subjects, and in the monitoring of isolated organs, such as a kidney, intended for transplantation.

1 . A sensor system for detecting creatinine in a sample, the system comprising:
 a) a composition comprising the enzymes creatininase, creatinase and sarcosine oxidase, respectively, at a ratio of between 10:5:1 and 49:8:1 U/ml, wherein the enzymes are in solution and the composition comprises a pH of 8.0-8.95;   b) at least a first amperometric sensor; and   c) a microfluidecs circuit adapted for maintaining a steady flow of a sample and a steady flow of said composition into said steady flow of sample to provide a reactio mix;   wherein the sensor system is arranged to allow said reaction mix to react for more than 60 seconds and less than 200 seconds before contact with said first amperometric sensor.   
     
     
         2 - 7 . (canceled) 
     
     
         8 . The sensor system according to  claim 1 , wherein the composition comprises a buffer selected from the group consisting of EPPS, HEPBS, POPSO, HEPPSO and MOBS. 
     
     
         9 . The sensor system according to  claim 1 , wherein the composition or the buffer is at a pH of 8.5. 
     
     
         10 . The sensor system according to  claim 1 , wherein the composition comprises EPPS at pH 8.0-8.5, optionally 50 mM EPPS at pH 8.0-8.5, optionally 50 mM EPPS at pH 8.0 or 50 mM EPPS at pH 8.5. 
     
     
         11 - 17 . (canceled) 
     
     
         18 . The sensor system according to  claim 1 , wherein the system further comprises means to take a sample, from a patient or a sample from a closed-loop isolated perfused kidney organ,
 wherein the sample from a patient is a microdialysate, from blood, urine, plasma, or tissue fluid.   
     
     
         19 - 32 . (canceled) 
     
     
         33 . A composition for detecting creatinine in a sample by converting creatinine into H 2 O 2  detectable by an amperometric sensor, the composition comprising the enzymes creatininase, creatinase, and sarcosine oxidase, respectively, at a ratio of between 10:5:1 and 49:8:1 U/ml, wherein all of the enzymes are in solution, and the composition comprises a pH of 8.0-8.95,
 wherein the composition is dilutable in a dialysate sample to provide a final mixed solution comprising more than 100 U/mol creatininase.   
     
     
         34 - 38 . (canceled) 
     
     
         39 . The composition of  claim 33 , wherein the composition comprises a buffer selected from the group consisting of EPPS, HEPBS, POPSO, HEPPSO and MOBS. 
     
     
         40 . (canceled) 
     
     
         41 . The composition according to  claim 33 , wherein the composition or a buffer is at a pH 8.5. 
     
     
         42 . The composition according to  claim 33 , wherein the composition comprises EPPS at pH 8.0-8.5, optionally 50 mM EPPS at pH 8.0-8.5, optionally 50 mM EPPS at pH 8.0 or 50 mM EPPS at pH 8.5. 
     
     
         43 - 53 . (canceled) 
     
     
         54 . A method for continuous monitoring of kidney function, said method comprising:
 a) providing a stream of isolated sample into a microfluidics circuit and maintaining a steady flow of the sample in said microfluidics circuit;   b) continuously challenging thesample in the microfluidics circit with a steady flow of a solution comprising creatininase, creatinase and sarcosine oxidase to provide a reaction mix comprising:
 i) a pH of 8.0-8.95; and 
 ii) creatininase, creatinase, and sarcosine oxidase, respectively, in solution at a U/mL ratio from 10:5:1 to 49:8:1 U/mL; 
   c) allowing said reaction mix to react for more than 60 seconds and less than 200 seconds before contact with an amperometric sensor, and detecting the level of H 2 O 2  in the reaction mix with said amperometric sensor, wherein creatininase, creatinase and sarcosine oxidase converts creatinine into H 2 O 2 , and   d) determining a creatinine clearance rate which is indicative of kidney function.   
     
     
         55 . (canceled) 
     
     
         56 . (canceled) 
     
     
         57 . The method according to  claim 54 , wherein the sample is a microdialysate. 
     
     
         58 . The method according to  claim 54 , wherein a creatinine clearance rate below a reference is indicative of impaired kidney function. 
     
     
         59 . The method according to  claim 54 , wherein the composition further comprises a buffer selected from the group consisting of EPPS, HEPBS, POPSO, HEPPSO and MOBS. 
     
     
         60 . The method according to  claim 54 , wherein the composition further comprises EPPS at pH 8.0-8.5, optionally 50 mM EPPS at pH 8.0-8.5, optionally 50 mM EPPS at pH 8.0 or 50 mM EPPS at pH 8.5. 
     
     
         61 . The sensor system according to  claim 1 , wherein the composition comprises a pH of 8.05-8.95; or 8.15-8.95. 
     
     
         62 . The composition according to  claim 33 , wherein the composition comprises a pH of 8.05-8.95; or 8.15-8.95. 
     
     
         63 . The method according to  claim 54 , wherein the composition comprises a pH of 8.05-8.95; or 8.15-8.95. 
     
     
         64 . The method according to  claim 54 , wherein the sample is taken from a subject. 
     
     
         65 . The method according to  claim 54 , wherein the sample is taken from an isolated perfused kidney.